The arenaviruses are a family of negative-sense RNA viruses that cause severe human disease ranging from aseptic meningitis to hemorrhagic fever syndromes. The arenavirus proteome consists of only four proteins: the small RING finger protein Z, nucleoprotein, viral RNA polymerase, and glycoprotein precursor, which is post-translationally modified to yield the heterodimeric envelope glycoproteins GP1 and GP2. While the functional role of these proteins for viral replication is well defined, their interactions with host cellular proteins, and the importance of these interactions for viral replication and disease pathogenesis, is largely unknown. Identification of novel arenavirus protein-host protein interactions that are critical for viral replication and/or viral pathogenesis would advance our understanding of the basic biology of these NIAID Category A viruses and provide new targets for the development of antivirals. Accordingly, we will use a cutting edge proteomics approach that features affinity purification of viral proteins in complex with host cellular proteins and multi-dimensional mass spectrometry protein identification technology (MudPIT) to identify host protein partners that interact with the proteomes of selected Old World (lymphocytic choriomeningitis virus) and New World (Junin virus) pathogenic arenaviruses. Our group has recently utilized this approach to successfully identify several host proteins that interact with viral proteins, including interactions that are conserved among both hantaviruses and arenaviruses. In addition, we have already generated the necessary reagents required for this work, namely a library of plasmid vectors that express, in mammalian cells, each of the ORFs encoded by six arenaviruses that are pathogenic for humans. The arenavirus protein-host protein interactions identified through this work will provide the basis for future grant applications to characterize these interactions more fully, particularly their impact on viral replication and pathogenesis. As an initial means to determine which interactions would be most relevant to pursue in future studies, we will evaluate the importance of the identified arenavirus protein-host protein interactions for viral replication by measuring viral replication in permissive cell lines following selective knock-down of host protein partners. PUBLIC HEALTH RELEVANCE: The arenaviruses cause severe human disease ranging from aseptic meningitis to hemorrhagic fever syndromes. These viruses produce four proteins. The goal of this project is to identify the human host proteins that these arenavirus proteins interact with during infection so that we can better understand how these virus protein-host protein interactions impact both the ability of the virus to replicate and cause human disease. Identification of important interactions will provide new targets for the development of antivirals.